大鼠脂肪源性干细胞与三维打印明胶支架的相容性

田晓红 张彬 房艳 柏树令* 敖强 佟浩

doi:10.16098/j.issn.0529-1356.2017.02.016

PDF(1044 KB)

解剖学报 ›› 2017, Vol. 48 ›› Issue (2) : 209-216. DOI: 10.16098/j.issn.0529-1356.2017.02.016

生物工程学

大鼠脂肪源性干细胞与三维打印明胶支架的相容性

田晓红¹张彬¹ 房艳² 柏树令^1* 敖强¹佟浩¹

作者信息 +

Compatibility between adipose derived stem cells of rats and three dimensional printing gelatin scaffold

TIAN Xiao-hong¹ ZHANG Bin¹ FANG Yan² BAI Shu-ling^1* AO Qiang¹ TONG Hao¹

Author information +

文章历史 +

摘要

目的通过观察大鼠脂肪源性干细胞（ADSCs）与三维打印（3DP）及戊二醛交联的明胶支架的相容性，筛选出最适合细胞生长的支架孔径，为进一步构建组织工程化组织或器官提供实验依据。方法采用酶消化法分离提取大鼠ADSCs，并用流式细胞术和多向诱导分化的方法进行鉴定；然后与不同孔径的3DP明胶支架复合培养，并用扫描电子显微镜和透射电子显微镜观察其超微结构，细胞活力分析仪检测其存活率；用MTT法检测二维（2D）与三维（3D）培养对ADSCs细胞活力的影响。结果获得的ADSCs具有多向分化潜能，具备干细胞的基本特征。接种ADSCs到3DP明胶支架后，扫描电子显微镜可看到细胞呈椭圆形或纺锤形，区别于传统二维培养的梭形形态；透射电子显微镜可看到细胞在支架空隙内散在分布，细胞核、细胞器等结构清晰，表明其与支架的相容性良好；90μm孔径的支架细胞存活率最高；3D培养的方法更有利于维持ADSCs的活力。结论 ADSCs与3DP明胶支架的相容性良好，90μm孔径的支架最适合ADSCs的生长。

Abstract

Objective To observie the compatibility of rat adiposederived stem cells (ADSCs) and three dimensional printing (3DP) gelatin scaffolds crosslinked with glutaraldehyde, the most suitable for cell growth aperture, in order to provide experimental basis for constructing tissue-engineered tissues or organs. Methods The enzyme digestion method was adopted to separate and extract rat ADSCs. Flow cytometry and multidirectional differentiation method were used for identification. The ADSCs were cultured with 3DP gelatin scaffolds of different aperturs. Scanning electron microscopy (SEM) and transmission electron microscope (TEM) were used to observe the ultra-microstructure. The cell vitality analyzer was used to detect its survival rate. MTT method was used to detect the effects of two dimensional (2D) and three dimensional (3D) cultural methods on ADSCs cell vitality. Results The seeding ADSCs had multi-directional differentiation potential, and had the basic characteristics of stem cells. After inoculation of ADSCs to 3DP gelatin scaffolds, cells were oval or spindle observed by SEM, which was different from the traditional 2D culture form. The cells scattered within the scaffold gap, the structures such as the nucleus and organelles were clear, which demonstrated a good compatibility with scaffold. The cell survival rate of 90μm aperture scaffold was the highest, and 3D cultural method was more advantageous to maintain the vitality of ADSCs. Conclusion The compatibility of ADSCs with 3DP gelatin scaffold is good, and 90μm aperture scaffold is most suitable for the growth of ADSCs.

导出引用

田晓红张彬房艳柏树令敖强佟浩. 大鼠脂肪源性干细胞与三维打印明胶支架的相容性[J]. 解剖学报. 2017, 48(2): 209-216 https://doi.org/10.16098/j.issn.0529-1356.2017.02.016

TIAN Xiao-hong ZHANG Bin FANG Yan BAI Shu-ling AO Qiang TONG Hao. Compatibility between adipose derived stem cells of rats and three dimensional printing gelatin scaffold[J]. Acta Anatomica Sinica. 2017, 48(2): 209-216 https://doi.org/10.16098/j.issn.0529-1356.2017.02.016

参考文献

［1］Gimble JM, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential ［J］. Cytotherapy, 2009, 5(5):362-369.
［2］Chia HN, Wu BM. Recent advances in 3D printing of biomaterials ［J］. J Biol Eng, 2015, 9(1):1-14.
［3］Castro NJ, Patel R, Zhang LG. Design of a novel 3D printed bioactive nanocomposite scaffold for improved osteochondral regeneration ［J］. Cell Mol Bioeng，2015，8(3)：416-432.
［4］Alemany-Ribes M, Semino CE. Bioengineering 3D environments for cancer models ［J］. Adv Drug Deliv Rev, 2014, 7980: 40-49.
［5］Zuk PA，Zhu M，Mizuno H，et al. Muhilineage cells from human adipose tissue：implications for cellbased therapies ［J］. Tissue Eng, 2001, 7(2)：211-226.
［6］Baer PC, Geiger H. Adipose-derived mesenchymal stromal/stem cells: tissue localization, characterization, and heterogeneity ［J］. Stem Cells Int, 2012, 2012：812693.
［7］Cawthorn WP, Scheller EL, MacDougald OA. Adipose tissue stem cells: the great WAT hope ［J］. Trends Endocrinol Metab，2012，23(6)：270-277.
［8］Keung EZ, Nelson PJ, Conrad C. Concise review: genetically engineered stem cell therapy targeting angiogenesis and tumor stroma in gastrointestinal malignancy ［J］. Stem Cells，2013，31(2)：227-235.
［9］Tian X, Fan J, Yu M. Adipose stem cells promote smooth muscle cells to secrete elastin in rat abdominal aortic aneurysm ［J］. PLoS One, 2014，9(9)：e108105.
［10］Wang JQ, Fan J, Gao JH, et al. Comparison of in vivo adipogenic capabilities of two different extracellular matrix microparticle scaffolds ［J］. Plast Reconstr Surg，2013, 131(2)：174e-187e.
［11］Fang Y, Tian X, Bai S, et al. Autologous transplantation of adipose-derived mesenchymal stem cells ameliorates streptozotocin-induced diabetic nephropathy in rats by inhibiting oxidative stress, pro-inflammatory cytokines and the p38 MAPK signaling pathway ［J］. Int J Mol Med, 2012, 30(1)：85-92.
［12］Wang JQ，Bai ShL，Hou WJ，et al. Isolation of human adipose-derived stem cells and the identification of biological characteristics ［J］. Acta Anatomica Sinica, 2011, 42(2)：205-210. (in Chinese)
王洁晴，柏树令，侯伟健，等.人脂肪源性干细胞的分离及生物学性状的鉴定［J］. 解剖学报，2011, 42(2)：205-210.
［13］Bai ShL, Gu XS, Zhang ChS. The Experiment Tutorial of Tissue Engineering ［M］. Beijing：People’s Military Medical Press, 2010：31-33. (in Chinese)
柏树令，顾晓松，张传森. 组织工程学实验教程［M］. 北京：人民军医出版社, 2010：31-33.
［14］Hao D, Duan XL, Bi XJ, et al. Isolation and culture of mouse adipose-derived stem cells and the expression of hematopoietic modulation factors before and after osteogenic induction ［J］. Journal of Clinical Rehabititative Tissue Engineering Research, 2012, 16(1): 51-55. (in Chinese)
郝丹，段显琳，毕晓娟，等. 小鼠脂肪源干细胞分离培养和成骨诱导前后造血调控因子的表达［J］. 中国组织工程研究与临床康复，2012, 16(1): 51-55.
［15］Wang Z, Nie J, Qin W, et al. Gelation process visualized by aggregation-induced emission fluorogens ［J］. Nat Commun, 2016, 7:12033.
［16］Pampaloni F, Reynaud EG, Stelzer EHK. The third dimension bridges the gap between cell culture and live tissue ［J］. Nat Rev Mol Cell Biol, 2007, 8(10):839-845.
［17］Mason C, Dunnill P. A brief definition of regenerative medicine ［J］. Regen Med, 2008, 3(1):1-5.
［18］Liu H, Fan H, Cui Y, et al. Effects of the controlled-released basic fibroblast growth factor from chitosan-gelatin microspheres on human fibroblasts cultured on a chitosan-gelatin scaffold［J］. Biomaeromoleeules，2007, 8(5):1446-1455.
［19］Puppi D, Chiellini F, Piras AM, et al. Polymeric materials for bone and cartilage repair ［J］. Prog Polym Sci, 2010, 35(4):403-440.
［20］Leong KF, Cheah CM, Chua CK. Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs［J］. Biomaterials, 2003, 24(13): 2363-2378.
［21］Hing KA, Best SM, Bonefield W. Characterization of porous hydroxyapatite［J］. J Mater Sci Mater Med, 1999, 10(3):135-145.
［22］Yan N, Zhang HD，Zhou L, et al. Scanning electron microscope analysis of structure of absorbable coral hydroxyapatite-produced by incomplete hydrothermal replacement reaction ［J］. The Journal of Practical Medicine, 2006, 22(18)：2086-2088. (in Chinese)
严宁，张汉东，周磊，等. Y500R可吸收珊瑚羟基磷灰石人工骨不完全热液置换反应前后结构的扫描电镜分析［J］.实用医学杂志，2006，22(18)：2086-2088.
［23］Cimetta E, Flaibani M, Mella M, et al. Enhancement of viability of muscle precursor cells on 3D scaffold in a perfusion bioreactor ［J］. Int J Artif Organs, 2007, 30(5): 415-428.
［24］Huang SB, Chou D, Chang YH, et al. Development of a pneumatically driven active cover lid for multi-well microplates for use in perfusion three-dimensional cell culture ［J］. Sci Rep, 2015, 5:18352.
［25］Mao XJ, Zeng Y, Han Q, et al. Gelatin microeryogels benefit human mesenchymal stem cells derived from adipose tissue to maintain stemness in vitro and promote application value in vivo ［J］. Basic and Clinical Medicine, 2015, 35(5):610-614. (in Chinese)
毛晓晶，曾洋，韩钦，等. 明胶微冰胶支架有利于脂肪来源间充质干细胞体外干性维持并提高其体内应用价值［J］. 基础医学与临床，2015，35(5)：610-614.